Protein kinases play a critical role in the regulation of virtually all aspects of cellular regulation and comprise one of the most active areas of research in the pharmaceutical industry today. The 522 protein kinase domains in the human genome may provide tremendous opportunities for developing new drugs for untreated disease, and the development of protein kinase inhibitors has increasingly become a major focus for the pharmaceutical industry. Protein kinase inhibitors have been reported to be useful in the treatment of numerous diseases including cancer, inflammatory and immunological diseases. See for example I. K. Mellinghoff and C. L. Sawyers, Kinase Inhibitor Therapy in Cancer, 14(12):1-11, 2000; J. Dumas, Growth factor receptor kinase inhibitors: recent progress and clinical impact, Current Opinion in Drug Discovery & Development, 4(4):378-89, 2001; J. Dumas, Protein kinase inhibitors: emerging pharmacophores, 1997-2000, Expert Opinion on Therapeutic Patents, 11(3):405-429, 2001; D. H. Williams and T. Mitchell, Latest developments in crystallography and structure-based design of protein kinase inhibitors as drug candidates, Current Opinion in Pharmacology, 2(5):567-73, 2002; S. B. Noonberg and C. C. Benz, Tyrosine kinase inhibitors targeted to the epidermal growth factor receptor subfamily: role as anticancer agents, Drugs, 59(4):753-67, 2000; S. Brunelleschi, L. Penengo, M. M. Santoro and G. Gaudino, Receptor tyrosine kinases as target for anti-cancer therapy, Current Pharmaceutical Design, 8(22):1959-72, 2002; P. G. Goekjian and M. R. Jirousek, Protein kinase C in the treatment of disease: signal transduction pathways, inhibitors, and agents in development, Current Medicinal Chemistry, 6(9):877-903, 1999; A. Gordon, The increasing efficacy of breast cancer treatment, Clinical Oncology (Royal College of Radiologists), 9(5):338-42, 1997.
While this large gene family represents a rich source of new drug targets, developing assays used to determine compound affinity can be highly problematic. Current high throughput screening assays for protein kinase inhibitors measure the incorporation of phosphate into a protein or peptide substrate. The most established method for assaying protein kinase inhibitors is a radiometric assay in which the gamma phosphate of ATP is labeled with either 32P or 33P. When the kinase transfers the gamma phosphate to the hydroxyl of the protein substrate during the phosphoryltransferase reaction the protein becomes covalently labeled with the isotope. The protein is removed from the labeled ATP and the amount of radioactive protein is determined. This assay is still the gold standard for quantitative protein kinase assays. Adaptation of this assay into a high throughput format is problematic due to the labor intensive separation steps and the large amounts of radioactivity that are used.
An alternative radiometric assay that is capable of higher throughput is the SPA or scintillation proximity assay (Amersham International). In this assay scintillant impregnated beads emit light when the labeled substrate is bound to the bead. This assay is limited by the level of radioactivity and the efficiency of the peptide substrate.
Techniques using fluorescence polarization to measure either protein kinase activity or inhibitor binding rely on a labeled antibody or peptide substrate. In these assays the enzyme transfers the gamma phosphate of ATP to a protein or peptide substrate. This activity is monitored by detecting the phospho-peptide by such means as an antibody. The binding of the antibody to the phospho-peptide will slow the free rotation of the peptide in solution and, therefore, a polarization signal from the product of the catalytic reaction can be detected. Examples include Burke et al., US 2001/0004522 A1 or T. C. Turek et al., Analytical Biochemistry, 299 (1), 25-53, 2001.
Many of the non-radioactive assays mentioned above use antibodies that recognize the product of the kinase reaction, i.e. a phospho-peptide. The binding assays use antibodies detected with enzyme-catalyzed luminescent readout. These methods are limited by reagent availability, well coating, and multiple wash incubation steps. Most importantly, however, antibody-based techniques for serine/threonine kinases require a specialized antibody for each kinase substrate. This requires that the phosphorylation site is known and that a antibody can be generated to that site. This increases the time, risk, and expense of assay generation. Additionally, only one phosphorylation site on a protein can be measured while, in practice, multiple sites on target proteins can be phosphorylated by a single kinase.
Another non-radioactive assay method utilizes the ATP-dependent activity of commercially available firefly luciferase. For example, U.S. Pat. No. 6,599,711 describes a method for measuring protein kinase activity by using bioluminescence to measure the change in ATP concentration following the phosphorylation of a protein kinase substrate in the presence of the protein kinase and ATP. The amount of light emitted by luciferase is directly proportional to the residual ATP following the kinase reaction. A tremendous advantage of this approach is that it does not require specialized detection reagents for each kinase thereby allowing screening panels to be set up in a single, common format.
We, and others, have shown that many protein kinases exhibit ATP hydrolysis in the absence of a protein acceptor substrate (New Tools for Screening Kinases: A Comparative Study”, The Society for Biomolecular Screening 9th Annual Conference, 2003, Portland, Oreg., Sep. 21-25, 2003, Kashem, M. A., Yingling, J., Nelson, R. M. and Homon, C. A.). In this case water acts as the terminal phosphate acceptor rather the hydroxyl of an amino acid as is traditionally the case in a phosphotransferase reaction. This ATPase activity is termed “intrinsic ATPase”. Assay formats based on ATP depletion will measure both phosphotransferase activity to protein and water. A drawback to the approach is that non-kinase ATPases that may be present as contaminants to the enzyme preparation will also show activity that could mistakenly be assigned to the protein kinase. Another potential complication is many protein kinases are assayed as enzymatic cascades wherein an upstream kinase phosphorylates and activates a downstream kinase. In a enzyme reaction in which multiple protein kinases are present, total ATP consumption is the sum of the activity of both the kinase of interest and the substrate kinase. Specific inhibition of the upstream kinase is likely to be obscured by the activity of the substrate kinase resulting in missed compounds which are active against the kinase of interest. Furthermore, if a kinase exhibited intrinsic ATPase activity equal or greater than its substrate-dependent phosphoryltransferase activity, identification of inhibitors that do not compete with ATP binding would not be possible. Very little has been published about the ATPase activity of protein kinases. In any case, it has never been considered a “problem” which anyone has tried to solve for quantification of ATP to determine kinase activity.